Altered regulation of the OmpF porin by Fis in Escherichia coli during an evolution experiment and between B and K-12 strains

Abstract

The phenotypic plasticity of global regulatory networks provides bacteria with rapid acclimation to a wide range of environmental conditions, while genetic changes in those networks provide additional flexibility as bacteria evolve across long time scales. We previously identified mutations in the global regulator-encoding gene fis that enhanced organismal fitness during a long-term evolution experiment with Escherichia coli. To gain insight into the effects of these mutations, we produced two-dimensional protein gels with strains carrying different fis alleles, including a beneficial evolved allele and one with an in-frame deletion. We found that Fis controls the expression of the major porin-encoding gene ompF in the E. coli B-derived ancestral strain used in the evolution experiment, a relationship that has not been described before. We further showed that this regulatory connection evolved over two different time scales, perhaps explaining why it was not observed before. On the longer time scale, we showed that this regulation of ompF by Fis is absent from the more widely studied K-12 strain and thus is specific to the B strain. On a shorter time scale, this regulatory linkage was lost during 20,000 generations of experimental evolution of the B strain. Finally, we mapped the Fis binding sites in the ompF regulatory region, and we present a hypothetical model of ompF expression that includes its other known regulators.

FIG. 1.

Effect of Fis level on OmpF expression in the E. coli B genetic background. (A) Amounts of OmpF in three strains that are isogenic except for their fis alleles. Porins were extracted from exponential-phase cultures after 4 h in DM250 medium for Anc, Anc_fis-1, and Anc_Δfis, which bear the ancestral, evolved, and deletion fis alleles, respectively, all in the Anc genetic background. Equal total amounts of protein (30 μg) were loaded on a gel, separated, and stained as described in Materials and Methods. The bands for OmpF, OmpA, and a standard nonspecific protein are shown. Reduced amounts of OmpF and increased levels of OmpA were associated with the lower levels of Fis in the two strains with mutant fis alleles; no effect on the nonspecific standard protein was observed. (B) Effect of Fis on transcription of ompF from its ancestral promoter. A single-copy chromosomal transcriptional fusion, PompF_Anc::lacZ, was constructed in the same three strains, Anc, Anc_fis-1, and Anc_Δfis, and in an evolved clone, Evol20K, isolated after 20,000 generations from population Ara-1. Cultures were grown in LB medium, and samples were taken during exponential growth (1 h, OD₆₀₀ = 0.1), transition to stationary phase (6 h, OD₆₀₀ = 3.7), and stationary phase (24 h, OD₆₀₀ = 3.2). The β-galactosidase specific activities were measured as μkat/mg protein (48) in three independent assays for each strain. Relative activities were then calculated using the value obtained for the Anc strain in exponential phase, with error bars showing 95% confidence intervals.

FIG. 2.

Binding of Fis to the ompF promoter region. (A) Electrophoretic mobility gel shift assays with an end-labeled DNA fragment spanning the PompF_Anc region from positions −422 to +148 relative to the transcription initiation site. The fragment was incubated with increasing concentrations of purified Fis (shown above the gel). The free DNA fragment is indicated by an arrow. (B) Specificity of Fis binding to the ompF promoter region. Electrophoretic mobility gel shift assays were performed with the same end-labeled DNA fragment and 20 nM Fis, both with and without unlabeled oligonucleotides as competitors. DNA (s) is a double-stranded 36-bp oligonucleotide containing high-affinity Fis-specific binding site I, present upstream of the fis promoter, while DNA (n) is a 34-bp nonspecific DNA where the Fis binding site was mutated (see Materials and Methods). The quantities of unlabeled DNA used in these experiments, ranging from 5- to 1,000-fold molar excesses, are shown above the gel (ng). Minus signs denote the absence of the corresponding component, while plus signs indicate the presence of Fis in the binding reaction mixture.

FIG. 3.

Mapping Fis-protected regions in PompF_Anc by DNase I footprint experiments. The entire promoter region was examined using four DNA fragments obtained by PCR amplification with the following primer pairs (shown on Fig. 4): ODS453-ODS452, ODS459-ODS452, ODS451-ODS453, and ODS458-ODS453. For each reaction, the first primer cited (shown above each gel) was radiolabeled prior to the PCR, except for ODS453-ODS452, where both primers were alternately labeled to generate footprints of both strands. The radiolabeled fragments were employed in footprint assays either in the absence (0) or presence of increasing concentrations of Fis (1, 10, and 40 nM, corresponding to the wedge above each gel). A Maxam-Gilbert reaction sequence was loaded in parallel (S) to map precisely the Fis-protected regions. These regions are indicated by bars to the left of each gel, and hypersensitivity sites are marked by asterisks on the right. The ompF transcriptional start site (+1) and translational start codon (ATG) are also shown.

FIG. 4.

Sequence of the ompF regulatory region in the E. coli B-derived Anc strain. The ompF promoter region is shown from positions −422 to +148 relative to the main transcriptional start site (represented by a bent arrow and an uppercase boldface letter). The −35 and −10 promoter boxes are indicated by dotted lines, and the ATG translational start site is shown in uppercase boldface letters with ompF above. Primers used to amplify DNA fragments for our experiments are shown above the sequence (ODS451 to -459), with arrowheads showing their 5′ to 3′ direction. The OmpR binding sites F1 to F4 (28) are indicated by thick solid lines above the sequence. Fis-protected regions I to V identified in this study are boxed when both DNA strands were protected in footprint experiments and marked with dashed lines when only one strand was protected. The filled circles under the sequence identify nucleotides identical to the consensus Fis binding site, Gnn(c/t)(A/g)(a/t)(a/t)(T/A)(t/a)(t/a)(T/c)(g/a)nnC (uppercase and lowercase indicate most conserved and less conserved residues, respectively) (21). The open circles and asterisks under the sequence indicate the DNase I hypersensitive sites for the Fis footprints of the DNA strand that is shown and its complement, respectively. Polymorphic sites that distinguish the B-derived Anc strain and K-12-derived CF7968 strain are shown in gray letters, with the nucleotide in CF7968 shown under the main sequence. The “t” indicates a single-base-pair indel in CF7968 compared to Anc, which places the adjacent “c” in the optimal position with respect to the consensus binding sequence for Fis.

FIG. 5.

Differential transcriptional regulation of ompF by Fis as a function of genetic context. (A) Effect of Fis on ompF transcription in K-12-derived strain CF7968. The transcriptional fusion PompF_CF::lacZ was introduced into the chromosomes of CF7968 and CF_Δfis. Cultures were grown in LB medium, and samples were taken in exponential growth (1, 1.5, and 2.5 h) and early stationary phase (6 h). The β-galactosidase specific activities were measured as μkat/mg protein (48) in three separate assays for each strain. Relative activities were calculated using the value for CF7968 at the first time point. Error bars show 95% confidence intervals. (B) Transcription of B and K-12 ompF promoter regions as a function of the B or K-12 genetic background. Transcriptional fusions PompF_Anc::lacZ and PompF_CF::lacZ were introduced into the chromosomes of the B-derived Anc and K-12-derived CF7968 strains. Cultures were grown in LB medium and sampled at the indicated time points. The β-galactosidase specific activities were measured as for panel A, again with three assays per strain. Relative activities were calculated from the value for the Anc-PompF_Anc::lacZ strain at the first time point. Error bars are 95% confidence intervals.

FIG. 6.

Model of ompF regulation in E. coli K-12 and B strains. (A) The “galloping” model for K-12 (87). (B) Our model for B-derived strains. Binding sites for OmpR-P and Fis are shown as open boxes labeled F1 to F4 and I to V, respectively. The −10 and −35 promoter boxes are shown as black boxes, and the transcriptional start site of ompF is marked by a bent arrow. OmpR-P proteins are shown as ovals, with a and b indicating the bound subsites. Fis dimers are shown as hexagons. Stars indicate the positions of the three mutational differences between the B-derived Anc sequence and the K-12-derived CF7968 sequence. When binding sites are bound by proteins, they are shown as thick lines and the protein is gray. When they are not bound, binding sites are shown as thin lines and the protein is white (OmpR binding is precluded by Fis in panel B). The number of Fis dimers bound to region I in the B-derived Anc strain is hypothetical and is inferred only from the size of the protected region.